1. Field of the Invention
Aspects of the present invention relate generally to the field of microscopy, and more specifically, to methods and apparatuses for dispensing immersion liquid for high resolution microscopy and lithography.
2. Description of Related Art
In a conventional manual optical microscope utilizing an immersion objective, the specimen substrate may be situated on top of a microscope stage with the sample located on the upper surface of the substrate (e.g., a slide) typically underneath a substrate cover (e.g., a cover slip). The microscope objective may be located directly above the substrate cover. Prior to viewing a specimen with a high numerical aperture objective, the microscopist may manually dispense a small quantity of the immersion liquid on to the surface of the specimen substrate cover above the specific region of the specimen to be viewed. To access the specimen substrate cover, the user may be required to move the objective lens away from the region of interest. After the fluid is applied, the user restores the immersion objective to the region of interest, and gradually approaches the sample, thereby trapping the liquid between the sample and the objective. While it is possible to flood the entire sample field of an upright microscope with an immersion liquid, this is rarely desirable in practical microscopy.
If the microscopist desires to manually scan or image a large area of the specimen, this may necessitate a manual reapplication or replenishment of the immersion liquid. As the microscopist moves either the microscope objective or the microscope stage relative to the other over the large area of the microscope substrate, immersion liquid may be lost, for example, by evaporation, by adhesion to previously scanned regions of the substrate, or by other means. On large scans, eventually the volume of immersion liquid may become insufficient to properly couple the rays to and/or from the front lens of the objective. In this case, the operator of a manual microscope will typically pause, move the objective, reapply fluid, and restore the objective. With short working-distance high-NA objectives this requires withdrawing the objective from the proximity of the sample, thereby losing focus.
In an otherwise automated microscope system, the need to replenish the immersion liquid frustrates the complete automation of the system. Current options for automatically replenishing immersion liquids are plagued with difficulties associated with keeping air bubbles from forming in the immersion liquid. For example, some of the prior art teaches incorporating ultrasonic devices into the microscope or replenishing system to aid in bubble removal.
The above described problems become more exacerbated when utilizing immersion objectives with inverted microscope systems in which the specimen being observed is situated between the user and the objective lens. In these systems, the specimen substrate may again be situated on top of a microscope stage with the sample located on the upper surface of the substrate, but, in an inverted system, the objective is positioned underneath the specimen substrate pointing up. If the microscopist is utilizing an immersion objective, the immersion liquid is typically deposited on to the surface of the upper-most lens of the immersion objective. Adding immersion liquid to the immersion objective therefore requires the ability to access to the top of the objective or alternatively to moving the objective into and out of the light path. Both methods may pose problems and limitation in the configuration of the inverted microscope and often result in restricting the possible travel of the optical system. Additionally, because the immersion liquid is typically applied directly to the objective lens, the inverted configuration increases the risk that liquid may enter the body of the objective. Sloppy microscopists frequently ruin expensive objectives by over-application of liquid. It should be noted that the above discussed problems relating to dispensing immersion liquids in viewing and imaging systems, also exists for high resolution lithography systems.
What is needed in the field is a method and apparatus that facilitates the viewing of specimens with immersion objectives that avoids the problems related to replenishment of the immersion liquid.